To achieve the long-term goal of restoring dental enamel, it is necessary to understand the fundamental chemical and biological principles of extracellular matrix assembly and the manner they control mineral nucleation and growth. There is still a large gap in our understanding of the underlying molecular mechanisms by which enamel matrix proteins assemble and interact with cells to control nucleation and oriented growth of hydroxyapatite crystals, and possibly cell movement and polarization. This is particularly true of ameloblastin protein, which is the focus of our proposed study. The goal of this proposal is therefore to advance our understanding of ameloblastin?s structure and function through a systematic investigation of its interactions with different targets. We hypothesize that the highly organized carbonated hydroxyapatite crystals in enamel continuously grow and form prismatic structures by means of complex ameloblastin-cell, ameloblastin- amelogenin, and ameloblastin-mineral interactions. Two major aims are proposed to systematically examine the above hypothesis by applying in vitro chemical models, cell culture and animal models for amelogenesis. Aim I: To investigate ameloblastin-cell membrane interactions and identify the interacting domains using in vitro synthetic liposomes and ameloblast-like cell culture model systems. We will design several mouse models with point mutations in the interacting domains identified, and examine the consequence of these mutations on enamel prismatic structure, ameloblast morphology, and the attachment of the cells to the matrix. We hypothesize that ameloblastin interacts with ameloblast cells via a domain in the sequence encoded by exon 5 and functions to anchor the mineralizing extracellular matrix to the enamel-forming cells affecting cell polarization, migration, and the formation of Tomes? processes. Aim II: To investigate ameloblastin-amelogenin interactions on the nanoscale in vivo and in vitro, and to identify their interacting domains using solution NMR spectroscopy. We will study the dynamics of calcium phosphate mineralization events on the nanoscale when ameloblastin is combined with amelogenin, using high resolution in situ atomic force (AFM) and Cryo- transmission electron microscopy (TEM). We hypothesize that amelogenin and ameloblastin form hetereomolecular entities that are functional during different stages of amelogenesis to control crystal formation. We anticipate to gain more insight into the structure, assembly properties and function of ameloblastin. We will define a novel cell- membrane- binding domain on ameloblastin protein. Novel protein- protein-interacting domains on the ameloblastin and amelogenin sequences will be identified. The effect of ameloblastin combined with amelogenin on mineralization will be elucidated and mineral-binding domains will be identified. These studies will advance understanding of the molecular function of ameloblastin in enamel biomineralization and will contribute to our efforts to fabricate synthetic enamel.